Our work in the area of mitochondrial function, energy homeostasis and metabolomics has led us to discover a remarkably strong association between adverse cardiometabolic outcomes and tissue/blood levels of acylcarnitine conjugates. These metabolites derive from acyl-CoA intermediates of fuel catabolism and permit mitochondrial export of excess carbons. Our working model positions acylcarnitines as biomarkers of mitochondrial stress and vehicles of stress relief. To test this hypothesis we have been studying the metabolic and physiological importance of carnitine acetyltransferase (CrAT), the mitochondrial matrix enzyme that converts acetyl-CoA and other short chain acyl-CoA species to their membrane permeant acylcarnitine counterparts. During the previous funding cycle we determined that the acyl group buffering capacity of CrAT is necessary for normal fuel selection, glucose control and exercise tolerance. One of the most exciting and potentially important discoveries we made is that genetic ablation of CrAT in mouse skeletal muscle increases tissue concentrations of acetyl-CoA and exacerbates diet-induced acetylation of mitochondrial proteins. Lysine acetylation (AcK) is reversible post-translational protein modification (PTM) in which a two carbon acetyl group is covalently bound to the e-amino group of a lysine residue. This PTM is found prominently on mitochondrial proteins and excessive AcK has been linked to metabolic disease in mice lacking sirtuin 3 (SIRT3), the principal mitochondrial-localized deacetylase enzyme that removes acetyl groups from specific lysine residues. Our preliminary data suggest AcK can occur non- enzymatically when the mitochondrial pool of acetyl-CoA expands. The proposed project applies state-of-the-art proteomics and metabolomics approaches to test our hypothesis that CrAT and SIRT3 function cooperatively to oppose mitochondrial carbon stress, and that coexisting insufficiencies in the function of these enzymes contribute to metabolic dysregulation in the context of metabolic disease. Because CrAT and SIRT3 depend on availability of essential micronutrient substrates, L-carnitine and nicotinamide, we will also determine whether a new combinatorial nutraceutical strategy that targets the two systems simultaneously might confer additive or perhaps synergetic metabolic benefits when administered to obese rodents.